EVIDENCE FOR UPTAKE OF VITAL DYE BY ACTIVATED RAT PERITONEAL MAST-CELLS - AN IN-VITRO IMAGING STUDY

Uptake of material from surrounding medium by activated rat peritoneal mast cells (PMCs) was studied using in vitro peritoneal eluate cells, the vital fluorescent dye sulforhodamine B (SFRM-B), secretagogue com pound 48/80, and an imaging technique. PMCs, which undergo different s tates of degranulation, are shown to possess the ability to take up (b y endocytosis) SFRM-B in an activity-dependent manner. The endocytosed dye is incorporated in the granules and can be discharged into the me dium when the cells are reactivated. Both the uptake and the discharge processes are calcium-dependent. The reactivity of mast cells to secr etagogue is not altered by the application of the dye. SFRM-B, a negat ively charged, nonspecific protein stain, displays greater photostabil ity and less leakage than the positively charged acridine orange, and its fluorescence persists for hours, whereas acridine orange fluoresce nce fades within 1 min when exposed to ultraviolet illumination. The f luorescent image of the dye-loaded mast cells can be preserved overnig ht in a container at room temperature. SFRM-B elicits no detectable da maging influence on the activated afferent discharge of splanchnic aff erent nerve fibers with mesenteric terminals. This enables the use of SFRM-B for studying the interactions between mesenteric afferent termi nals and their surrounding mast cells. (C) 1994 Academic Press, Inc.